Mucin immobilized chromatography

ABSTRACT

An in vitro high-throughput screening method that models the absorption of drugs across the epithelial mucosa is provided. The invention discloses an in vitro mucin immobilized chromatography model to estimate drug permeability coefficients. The invention describes compositions for mucin immobilized chromatography media and methods to prepare this media. The invention also discloses a method to estimate in vitro drug permeability coefficients using mucin immobilized chromatography media. The invention also discloses methods to determine absorption processes in the digestive system and discloses methods of use for mucin chromatography media in column, batch, assay, diagnostic, or high-throughput screening analyses.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/348,184, filed Jan. 14, 2002, the content of which isincorporated by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention relates to an in vitro mucin immobilizedchromatography (MIC) model to estimate drug permeability coefficient.More particularly, the present invention provides compositions for MICmedia and methods for preparing MIC media. In addition, the inventionprovides methods to estimate in vitro drug permeability coefficientusing MIC media, including, inter alia column or batch chromatography orhigh-throughput screening (HTS) analyses. The present invention furtherrelates to methods for estimating drug permeability coefficients inindividuals, including healthy and diseased individuals using the invitro MIC model. The instant invention also relates to methods foremulating absorption processes in the digestive system and in othertissues and organs comprising a mucosal layer.

BACKGROUND OF THE INVENTION

[0003] Oral drug administration is a noninvasive route of drug deliveryfor the treatment or prevention of diseased states in animals. Thesuccess of oral drug administration depends upon many factors, one ofwhich is the degree of bioavailability of the drug. Bioavailabilityaccounts for “the rate and extent to which the active ingredient oractive moiety is absorbed from a drug product and becomes available atthe site of action.” 21 C.F.R. §320.1 (2000). In general, a host ofbiological factors affect bioavailability, including pharmacokineticbehavior of the drug, drug formulation, site of administration,formulation and dosage form, and the physiological state of the patient.

[0004] In oral drug administration, one of the critical steps indetermining bioavailability is the transportation and absorption ofdrugs across the various cell membranes comprising the intestinal mucosa(Lennernäs, H., “Human Perfusion Studies” in Oral Drug Absorption:Prediction and Assessment, Dressman, J. and Lennernäs, H., Eds., MarcelDekker, 106, 99-117 (2000)). An important variable in calculating theextent of absorption is the effective permeability, P_(eff), becauseP_(eff) is the rate-limiting step in the absorption process (Stein, W.D., Transport and Diffusion across Cell Membranes, Academic Press, Inc.,Orlando, Fla., 1986).

[0005] Drug transport proceeds either by carrier-mediated transport,transcellular transport or by passive diffusion mechanisms. Mostorally-administered drugs are absorbed by passive diffusion mechanisms.For drugs that are absorbed by passive diffusion, there are a number ofin vivo and in vitro intestinal absorption models that can be used toestimate the absorption of potential oral drug candidates during initialdrug screening tests.

[0006] In order to accurately measure P_(eff), Lennernäs et al.developed an in vivo human single-pass intestinal perfusion model tomeasure membrane transport. A large number of drugs belonging to severalpharmacological classes were measured using this model, includingcardiovascular system agents, nonsteroidal anti-inflammatory drugs(NSAIDs), central nervous system (CNS) agents, anti-infective agents,urinary tract system agents, and gastrbenterological agents. These dataserve as a guideline for classifying drug substances based on theiraqueous solubility and intestinal permeability under theBiopharmaceutical Classification System (BCS)(http://www.fda.gov/cder/guidance/3618fnl.htm). The in vivo datagenerated by this method provide accurate P_(eff) values, and are usefulfor comparative studies with in vitro data; however, the measurement ofbioavailability by the human intestinal perfusion model or by other invivo methods is impractical for HTS.

[0007] To this end, a number of in vitro absorption models have beendeveloped to calculate and predict the parameters involved in drugabsorption more quickly than in vivo models. These include the isolatedintestinal cell model (Osiecka et al., Pharm. Res. 2, 284-293 (1985)),the everted intestinal ring model (Leppert and Fix, J. Pharm. Sci. 83,976-981 (1994)), the everted intestinal sac model (Barthe et al., J.Drug Metab. Pharmocokinet. 23, 313-323 (1998)), the Ussing chamber model(Ussing and Zerahn, Acta. Phisiol. Scand. 23, 110-127 (1951)), theoctanol-water partitioning model (Fujita et al., J. Am. Chem. Soc. 86,5175-5180, (1964)), and the Caco-2 cell model (Artursson and Karlsson,Biochem. Biophy. Res. Comm. 175, 880-885 (1991)). Other modelsspecifically utilizing chromatography methods-include octadecyl-reversedphase chromatography (ODS) (Dorsey and Khaledi, J. Chromatography A 656,485-499 (1993)), immobilized artificial membranes (IAM) (Yang et al.,Advanced Drug Delivery Reviews 23, 229-256 (1996);http://www.registech.com/iam/index.htm), and micellar liquidchromatography (MLC) (Molero-Monfort et al., J. Chromatography A 870,1-11 (2000)).

[0008] The accuracy of correlation of in vitro drug absorption values tothose obtained by in vivo methods varies according to the model used.For example, one widely used in vitro model, the human intestinal Caco-2cell line, emulates the intestinal cellular epithelium in humans verywell (Artursson and Karlsson, Biochem. Biophy. Res. Comm. 175, 880-885(1991)). Other in vitro models that may be used include the humanintestinal epithelial cell lines, HT29-H and a co-culture of Caco-2 andHT29-MTX, wherein the outer surface of the cells contain a secretedmucin layer (Wikman et al., Pharmaceutical Research 10, 6, 843-852(1993); Hilgendorf et al., J. Pharm. Sci. 89, 1, 63-75 (2000)). However,the experimental use of cell lines require costly and continuousmaintenance programs to ensure cell viability. As a result, highperformance liquid chromatography (HPLC) models, such as immobilizedartificial membranes (IAM) and micellar liquid chromatography (MLC),which are experimentally easier to use than cell culture models, havebeen developed and shown to predict drug absorption with reliability andaccuracy comparable to that of the Caco-2 cell model (Stewart et al.,Pharm. Res. 15, 1401-1406 (1998)).

[0009] IAM creates mechanically stable chromatographic surfaces thatmimic cell membranes (Pidgeon et al., Applications of EnzymeBiotechnology, 201-220 (1991)). The cell membrane structures are modeledafter liposomes (Bangham et al., Methods Membr. Biol. 1, 1-68 (1974))and created by the covalent attachment of membrane-forming lipids,largely phospholipids, to a chromatographic surface.

[0010] U.S. Pat. No. 4,927,879 discloses a method for forming an IAM bythe covalent attachment of amphiphilic cyclic dicarboxylic anhydrides tosilica. The attached molecules form a tightly packed arrangement on thesurface of the support to prevent additional nucleophilic reactions tooccur at these sites. U.S. Pat. No. 4,931,498 describes compositions andmethods used to immobilize the membrane-forming lipids to silicasupports.

[0011] Unlike IAM models, MLC models comprise a reversed stationaryphase in combination with a surfactant solution mobile phase, where inthe surfactant concentration in the mobile phase exceeds its criticalmicelle concentration (CMC) (Escuder-Gilabert et al., J. ChromatographyB 740, 59-70 (2000)). The surfactant, polyoxyethylene lauryl ether(Brij-35), adsorbs to the hydrophobic stationary phase and behaves asthe polar membrane region of the cell. If supplemented with saline, theBrij-35 mobile phase further acts as an extracellular fluid. Like IAMmodels, MLC models predict the partitioning behavior of small moleculeabsorption into the lipid bilayers of the cell membrane.

[0012] IAM and MLC are currently being used to predict oral drugabsorption and bioavailability. However, IAM and MLC methods are limitedbecause they only model one part of the absorptive process, that is, thepassive diffusion across the lipid bilayer. Neither IAM nor MLC modelsthe initial absorption process across the epithelial mucosa, a substancethat is critical for drug absorption.

[0013] The epithelial mucosa, or mucus, is a hydrogel comprising interalia, mucins, lipids, proteins, DNA, RNA and carbohydrates. Theepithelial mucosa covers all epithelial surfaces, including respiratory,buccal, gastrointestinal, reproductive and urinary tract surfaces andcoats both the plasma membrane. Mucus functions to protect the cellsurface from harmful extracellular molecules and to regulate cellularinteractions and molecular uptake that occur between the cell and itsenvironment (Braybrooks et al., J. Pharm. Pharmac. 27, 508-515 (1975);Larhed et al., J. Pharm. Sci. 86, 660-665 (1997); Larhed et al., Pharm.Res. 15, 66-71(1998)).

[0014] Mucins, major components of mucus, are a family of high molecularweight glycosylated proteins that impart viscous and viscoelasticproperties on mucus (Strous and Dekker, Critical Reviews in Biochemistryand Molecular Biology, 27, 57-92 (1992)). Differences in type, level,and pattern of glycosylation in mucins is often altered in certaindiseased states, including cancer (see, e.g., (Bhavanandan, Glycobiology1, 493-503 (1991); Finn et al., U.S. Pat. No. 5,827,666). Mucins areprimarily responsible for determining whether a substance, such as adrug, crosses the mucosal layer to enter the cells underlying themucosal layer.

[0015] To date, in vitro models fail to provide a fast and convenientmeans to emulate the outer layer of the cell membrane and the cellularmucosa in order to investigate drug absorption. It would therefore bedesirable to develop a chromatography model that measures the initialabsorption process of a drug across a mimetic mucosal surface in orderto obtain a more accurate representation of drug absorption acrossepithelial mucosa. The would be particularly useful for modelinggastrointestinal drug absorption after oral administration. In addition,it would be desirable to develop a fast, cost efficient method toestimate drug absorption for HTS in both healthy and diseased states.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to an in vitro mucinimmobilized chromatography model (MIC) that may be used to determinedrug permeability coefficients across mucosal membranes. The inventionsimulates the initial step in the fate of the drug, the interaction ofthe drug with mucin components and cell membrane of the epithelialmucosa.

[0017] The instant invention provides compositions for MIC media andmethods for preparing MIC media. In addition, the invention providesmethods to estimate in vitro drug permeability coefficients using MICmedia, including, inter alia column or batch chromatography or HTSscreening analyses. The present invention further provides methods forestimating drug permeability in individuals, including healthy anddiseased individuals using the in vitro MIC model. The instant inventionalso relates to methods for emulating absorption processes in thedigestive system and in other tissues and organs comprising a mucosallayer.

[0018] The MIC media comprise a mucin-type protein or a mucin-typepeptide non-covalently immobilized to a solid support matrix, whereinsaid solid support matrix is a surface modified with an amphiphlicmolecule. The MIC media may comprise one or more different types ofmucin-type proteins or mucin-type peptides. The MIC media may furthercomprise one or more components of the mucus layer, including a lipid, anon-mucin protein, DNA, RNA, or a carbohydrate. According to anotherembodiment of the instant invention, the composition may comprise both amucin-type protein and a mucin-type peptide. The mucin-type protein ormucin-type peptide may be derived from a secreted mucin or amembrane-bound mucin. The MIC media may comprise a combination ofsecreted and membrane-bound mucins.

[0019] The mucin-type proteins of the MIC media may have a variety oftypes and levels of glycosylation. The mucin-type protein may containthreonine and serine amino acid residues that are fully O-linkedglycosylated, partially O-linked glycosylated, or non-glycosylated atthe hydroxyl group of each serine or threonine amino acid side chain.The hydroxyl group may be glycosylated with a carbohydrate selected fromthe group consisting of monosaccharides, disaccharides,oligosaccharides, and polysaccharides. Preferably, an oligosaccharide isselected from the group consisting of Gal β(1-3)-GalNAc α(1-0)-, Galβ(1-3)-[GlcNAc β(1-6)]-GalNAc α((1-0)-, GlcNAc β(1-3)-GalNAc α(1-0)-,GlcNAc β(1-3)-[GlcNAc β(1-6)]-GalNAc α-(1-0)-, GalNAc α(1-3)-GalNAcα-(1-0)-, and Gal β(1-3)-[Gal β(1-6)]GalNAc α(1-0)-. In anotherpreferred embodiment, a disaccharide is Galβ(1-3)GalNAc α-. In anotherpreferred embodiment, a monosaccharide is GalNAc.

[0020] Similarly, mucin-type proteins may be N-linked glycosylated atasparagine amino acid residues found in the protein chain. Themucin-type protein may contain asparagine amino acid residues that arefully N-linked glycosylated, partially N-linked glycosylated, ornon-glycosylated at each amide group of asparagine that is part of thesequence of amino acids Asn-X-Ser or Asn-X-Thr, wherein X is any aminoacid other than proline and aspartic acid. The amine group may beglycosylated with a carbohydate selected from the group consisting ofmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.Preferably, the N-linked side chain is glycosylated with ahexasaccharide, GlcNAc₃Man₃.

[0021] In another aspect of the present invention, the mucin-typepeptide is non-glycosylated. In another embodiment, the mucin-typepeptide, whether glycosylated or not, is an epitope associated withmucin from either malignant or normal cells.

[0022] This invention also relates to a MIC composition wherein themucin-type protein or mucin-type peptide is derived from the epithelialcell surface coatings of the gastrointestinal (GI) tract, mouth, eye,trachea, lungs, salivary glands, sweat glands, breast, reproductivetract (e.g., vagina), pancreatic duct, gall bladder or urinary tract(e.g., urethra or vagina). The mucin-type protein or mucin-type peptidemay be derived from normal or cancerous epithelial cells. Anotherembodiment of the instant invention is a MIC composition wherein themucin-type protein or mucin-type peptide is derived from thetransmembrane domain of mucins from epithelial cells of the GI tract,mouth, eye, trachea, lungs, salivary glands, sweat glands, breast,reproductive tract, pancreatic duct, gall bladder or urethra.

[0023] In a further embodiment, the instant invention relates to an MICmedia that comprises a mucin-type protein or mucin-type peptide that isderived from a mammalian mucin. Said mammalian mucin may be selectedfrom the group consisting of human, ape, monkey, rat, pig, dog, rabbit,cat, cow, horse, mouse, rat and goat. In yet a further embodiment, thepresent invention relates to a MIC media comprising a mucin-type proteinor mucin-type peptide that comprises at least one tandem repeat sequenceselected from the group consisting of SEQ ID NOS:1-12. In anotherembodiment, a mucin-type peptide is 5 to 50 amino acids in length and amucin-type protein is greater than 50 amino acids in length.

[0024] In another embodiment, the invention provides an MIC mediacomprising a solid support matrix that comprises an inorganic molecule,a polymer, or a copolymer. The inorganic molecule may be selected fromsilicates, aluminas, hydroxyapatites, zeolites, germanates, phosphatesor a mixture thereof. In a preferred embodiment, the inorganic moleculeis a functionalized silicate. In a more preferred embodiment, thefunctionalized silica gel is selected from the group consisting of3-aminopropyl silica, 1-allyl silica, 3-(3,4-cyclohexyldiol)propyl,3-(diethylenetriamino)propyl, 4-ethyl benzenesulfonamide,3-mercaptopropyl, propionyl chloride, 3-(2-succinic anhydride)propyl,and 3-(ureido)propyl.

[0025] In another preferred embodiment, the solid support matrix is apolymer selected from the group consisting of agarose, dextran,polystyrene, polyvinyl alcohol, polymethylacrylate,polymethylmethacrylate and acrylamides. In another preferred embodiment,the solid support matrix is a copolymer selected from the groupconsisting of polyethyleneglycol-co-polystyrene,polystyrene-co-divinylbenzene, sulfonated styrene-divinylbenzene,polyvinylchloride-co-vinylacetate, bis-acrylamide-azalactone,N-vinylpyrrolidone-co-divinylbenzene, polyethylene-co-butylmethacrylate, poly(lactic-co-glycolic acid), lignin-cresol copolymers,dextran-agarose copolymers, glycidyl methacrylate-ethylenedimethacrylate copolymers, poly(lactide-co-caprolactone) and polyacryliccopolymers.

[0026] In another aspect, the invention provides a MIC media thatcomprises an amphiphilic molecule that is covalently attached to thesurface of a solid support matrix. In one embodiment, the moleculecomprises an epithelial cell membrane constituent. In a preferredembodiment, the amphiphilic molecule is selected from the groupconsisting of phospholipids, phosphoglycerides, spingolipids,prostaglandins, saturated fatty acids, unsaturated fatty acids, andsoaps. Preferably, the amphiphilic molecule is a phospholipid. In a morepreferred embodiment, the amphiphilic molecule is phosphatidyl choline.

[0027] The present invention provides a MIC media in which themucin-type proteins and mucin-type peptides are prepared by isolationfrom a biological source or by recombinant DNA methods, syntheticchemical routes, or a combination of any of these methods.

[0028] Recombinant DNA methods include expressing the protein or peptideof interest from a host cell that comprises a nucleic acid moleculecomprising a nucleic acid sequence encoding the protein or peptide ofinterest. The host cell may comprise a vector comprising the nucleicacid sequence. The vector may further comprise an expression controlsequence operably linked to the nucleic acid sequence. In a preferredembodiment, the nucleic acid sequence encodes an amino acid sequenceselected from the group consisting of a mucin-type protein and amucin-type peptide. The mucin-type peptide may comprise a mucin epitope.

[0029] The host cell may be a prokaryotic or a eukaryotic cell. In apreferred embodiment, the prokaryotic cell is an E. coli cell. Inanother preferred embodiment, the eukaryotic cell may be selected fromthe group consisting of yeast, insect, and mammalian cells.

[0030] Another aspect of the present invention is to provide a methodfor the preparation of a chromatography medium. The method comprisesproviding a solution of a mucin-type protein or peptide and mixing thesolution with a solid support matrix that is surface modified with anamphiphilic molecule under conditions in which the protein or peptideforms a non-covalent interaction with the matrix. In a preferredembodiment, the mucin-type protein or mucin-type peptide may bedissolved in a solvent. In a more preferred embodiment, the solvent maybe acetone, Dulbecco phosphate buffered saline (DPBS) optionallycomprising a non-ionic surfactant such as Brij-35. The solvent may beany other suitable solvent as well. The mixture may be washed and/ordried before use. In one embodiment, the mixture is dried byevaporation. The evaporating step may be evaporation by air or byrotoevaporation, wherein both heat and vacuum are applied.

[0031] The MIC media may be used in any method, including, inter alia,batch chromatography or column chromatography, which includes liquidchromatography, high performance liquid chromatography, and fastperformance liquid chromatography. The MIC media may also be used tocoat a slide or plate for thin layer chromatography. In anotherembodiment, the MIC media may be used to coat a multi-well plate for HTSor other high-throughput analyses.

[0032] Another aspect of the present invention is to provide in vitromethods for estimating drug absorption through the mucus layer. Thismethod comprises determining an effective permeability coefficient(P_(eff)) for a drug compound using MIC media.

[0033] In one aspect, the invention provides a method of estimating thepermeability coefficient of one or more drugs by contacting the drugwith the MIC media and measuring the degree of binding of the drug onthe MIC media and estimate the drug permeability coefficient. In apreferred embodiment, the contacting step is performed by loading thedrug on a column comprising the MIC media and the measuring step isperformed by measuring the retention time of the drug on the column. Inone embodiment, the drug permeability coefficient is estimated for asingle drug. In another preferred embodiment, permeability coefficientsof a number of drugs are measured either sequentially, by contactingeach drug with the MIC media one by one and measuring binding of thedrug to the media, or simultaneously, by contacting a mixture of morethan one drug with the MIC media and measuring binding of the drug tothe media. In another embodiment, the MIC media comprises types and/oramounts of mucin-type protein or mucin-type peptide to mimic the mucuslayer in a specific cell, tissue or organ. These include the mucus layerof the GI tract, eye, trachea, lungs, salivary glands, sweat glands,breast, reproductive tract, pancreatic duct, gall bladder or urinarytract.

[0034] In another aspect, the method may be used to compare the relativepermeability of one or more drugs to each other or to drugs having aknown permeability. The method comprises contacting the MIC media with adrug of interest and measuring the degree of binding of the drug to theMIC media, then comparing the degree of binding to that measured for oneor more other drugs. The other drug may be one having an unknown drugpermeability coefficient or may be one having a known drug permeabilitycoefficient. The method may be used to determine whether the drug ofinterest has a greater or lesser relative permeability compared to theother drug. Further, by comparing the drug of interest to one or moreother drugs having a known permeability coefficient, one may estimatethe permeability coefficient for the drug of interest. The degree ofbinding may be measured sequentially or simultaneously. The degree ofbinding may be determined using column chromatography or by batchchromatography. See Example 5.

[0035] Another embodiment of the invention is to provide a method forestimating effective permeability coefficients of one or more drugs intwo or more physiological states by changing the amount and/or type ofmucin-type protein or mucin-type peptide in the MIC media. In onepreferred embodiment, the MIC media comprises types and/or amounts ofmucin-type proteins or mucin-type peptides that mimic the mucus layer ina healthy state or in a diseased states. In another embodiment, themethod provides a method for estimating effective permeabilitycoefficients of one or more drugs in two or more physiological states bychanging the mobile phase of the media. In a preferred embodiment, thepH, osmolarity and/or surfactant concentration or type is altered tomimic a particular physiological state.

[0036] The MIC media may be used to mimic the healthy or diseased stateof any mucosal layer, including the gastrointestinal tract, reproductivetract, urinary tract, eye, mouth, salivary glands, pancreatic glands,sweat glands or gall bladder. In a preferred embodiment, the diseasedstate is epithelial cell cancer.

[0037] In another embodiment, the different physiological states aredifferent developmental stages, e.g., infant, child, adolescent andadult. In another embodiment, the different physiological states may bemucosal layers from different cells, tissues, or organs. For example,one may compare estimated drug absorption in the mouth, gastrointestinaltract and reproductive tract to determine the best method ofadministration.

[0038] A further embodiment of this invention is to provide a method foremulating an absorption process in a system comprising more than oneepithelial mucosal layer. The method comprises estimating drugpermeability coefficients using a series of chromatography columns eachhaving a different amount and/or type of a mucin-type protein,mucin-type peptide, or combination thereof immobilized to achromatography medium, wherein the different amounts and/or types ofmucin-type proteins or mucin-type peptides mimic the different mucuslinings in the system. In a preferred embodiment, the mobile phase maybe altered to mimic the different extracellular fluids in the system. Ina preferred embodiment, the system is the digestive system.

[0039] Another embodiment of the instant invention is a kit comprisingthe MIC media. The kits may be used to estimate absorption of one ormore drugs.

[0040] Another embodiment of the instant invention comprises a methodfor using MIC in HTS.

BRIEF DESCRIPTION OF DRAWINGS

[0041]FIG. 1 illustrates an optimization plot between the amount ofmucin absorbed to IAM beads versus the weight ratio of mucin to IAMbeads. See Example 1.

[0042] FIGS. 2(a) and 2(b) show a transmission electron micrograph (TEM)of MUC-SAC to IAM particles

[0043]FIG. 2(c) shows a TEM of IAM beads without mucin. See Example 2.

[0044] FIGS. 3(a) and 3(b) show confocal micrographs and FIG. 3(c) showa phase contrast micrograph of MUC5AC-IAM particles. Anti-MUC5ACantibody was labeled with biotin and visualized with streptavidinlabeled with Alexa Fluor 488. See Example 3.

[0045]FIG. 4 illustrates the thickness of the mucin-phosphatidyl cholinelayer on the solid matrix as measured by confocal microscopy. SeeExample 3.

[0046]FIG. 5 illustrates the correlation between P_(eff Loc I-Gut) andk′_(MIC). See Example 5.

[0047]FIG. 6 shows the correlation between the measured partitioncoefficient determined by MIC and the membrane equilibrium constantderived from equation 2, using D_(m) and P_(eff MIC) values listed inTable 4 and L=0.36 μm.

[0048]FIG. 7 shows the correlation between in vivo percent drugabsorption with the Peff found by the MIC, Caco-2, and Loc-I-Gutmethods. See Example 5.

[0049]FIG. 8 shows the correlation between in vivo absorption valueswith k′_(IAM) and k′_(MIC). See Example 5.

[0050]FIG. 9 shows the correlation between P_(eff Loc-I-Gut) andk′_(MIC(2:1)) and k′_(MIC(1:2)). See Example 7.

DETAILED DESCRIPTION OF INVENTION

[0051] Definitions

[0052] Unless otherwise defined herein, scientific and technical termsused in connection with the present invention shall have the meaningsthat are commonly understood by those of ordinary skill in art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, columnchromatography, recombinant DNA methods, peptide and proteinchemistries, and conjugation chemistries described herein are those wellknown and commonly used in the art. The methods and techniques of thepresent invention are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates(1992, and Supplements to 2002); Hermanson, Bioconjugate Techniques,Academic Press, San Diego, Calif. (1996); Solid-Phase Peptide Synthesis,G. B. Fields, Ed., 1st ed., Academic Press, New York, 289 (1997);Handbook of Chromatography: General Data and Principles Volume II, G.Zweig and J. Sherma, Eds., CRC Press, Boca Raton, Fla. (1972); each ofwhich is incorporated herein by reference in its entirety.

[0053] The following terms, unless otherwise indicated, shall beunderstood to have the following meanings:

[0054] Chromatography medium is defined as either a liquid or a solidstationary phase used in separation techniques.

[0055] Solid support matrix is any solid stationary phase comprised oforganic or inorganic molecules, polymers, copolymers or combinationsthereof.

[0056] An inorganic molecule as used herein refers to any suitableinorganic molecule that may be used as a solid support matrix for MICmedia. In a preferred embodiment, the inorganic molecule is selectedfrom silicates, aluminas, hydroxyapatites, zeolites, germanates orphosphates.

[0057] A polymer refers to a molecule having a molecular weight ofapproximately 5,000 or greater, which is composed of monomer units ofless than 5,000 molecular weight covalently bonded together.

[0058] The term copolymer comprises a polymer having more than onemonomer unit per chain. The sequence of monomer units within the overallcomposition of a copolymer can be alternating, block, or statistical(Odian, Principles of Polymerization, 3rd Ed., 142-149 (1991)).

[0059] An amphiphilic molecule is a molecule having a polar domain and anonpolar domain.

[0060] The term mucus layer is defined as a lining on an epithelial cellsurface of the GI tract, eye, trachea, lungs, salivary glands, sweatglands, breast, reproductive tract, pancreatic duct, gall bladder orurinary tract. The mucus lining may be derived from a normal cell,tissue or organ or from a cancerous cell, tissue or organ. The mucuslayer is comprised of a number of components including mucins, lipids,proteins, DNA, RNA and carbohydrates.

[0061] The term glycoprotein is defined as a molecule comprising acarbohydrate moiety and a proteinaceous moiety. A comprehensive databaseof glycoproteins and their O-linked glycosylation sites from a varietyof species is available athttp://www.cbs.dtu.dk/databases/OGLYCBASE/Oglyc. base.gz.

[0062] Mucin refers to a class of high molecular weight glycosylatedproteins that are either secreted or membrane-bound and that form aviscous gel that cover epithelial cells. Secreted mucins refer toproteins that are not kinked via a transmembrane region to the cells.They are synthesized by epithelial cells that are stored intracellularlyprior to being transported to the apical side of the cells where theyare released into the extracellular space to form a part of the mucuslayer. Secreted mucins form oligomeric, crosslinked molecular aggregatesor viscoelastic gels. Membrane-bound mucins refer to integral membraneproteins synthesized in epithelial cells that are not storedintracellularly prior to being transported to apical plasma membrane.Membrane-bound mucins are typically monomeric and contain proteindomains associated with signaling processes.

[0063] Glycosylation refers to the process of adding carbohydratemoieties to protein side-chains. O-linked glycosylation occurs at thehydroxyl group of threonine or serine and N-linked glycosylation occursat the amino group of asparagine. Both O-linked and N-linkedcarbohydrate moieties can be monosaccharides, disaccharides,oligosaccharides, or polysaccharides. A monosaccharide may be either analdose or a ketose and is classified as a triose, tetrose, pentose,hexose, or heptose depending upon the number of carbons in thecarbohydrate moiety. Examples of monosaccharides include glyceraldehyde,ribose, xylose, arabinose, glucose, galactose, mannose, ribulose,fructose, glucopyranose, ribofuranose, and fructofuranose. Adisaccharide is two covalently linked monosaccharides. Examples ofdisaccharides include maltose, lactose, and sucrose. An oligosaccharideis defined as 3-9 covalently-linked monosaccharides, which are O-linkedor N-linked to each other. A polysaccharide is defined as a polymer ofapproximately greater than about ten monosaccharide residues linkedglycosidically in branched or unbranched chains. Examples of apolysaccharide include amylose, amylopectin, glycogen, cellulose, andstarch.

[0064] A mucin-type protein or mucin-type peptide is fully O-linkedglycosylated when each serine and threonine residue in the tandem repeatregion in a specific mucin-type protein molecule or mucin-type peptidemolecule is O-linked glycosylated. Similarly, a mucin-type protein ormucin-type peptide is fully N-linked glycosylated when each asparagineresidue in the tandem repeat region and in the consensus sequence ofamino acids Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid otherthan proline and aspartic acid, is N-linked glycosylated. A mucin-typeprotein or mucin-type peptide is partially O-linked glycosylated whenbetween 10-90% serine and threonine residues are O-linked glycosylatedin the tandem repeat region or when between 10-90% asparagine residuesare N-linked glycosylated in the tandem repeat region. A mucin-typeprotein or mucin-type peptide is non-glycosylated when no serine orthreonine residue is O-linked glycosylated or when no asparagine residueis N-linked glycosylated.

[0065] A peptide is defined as two to 50 amino acids and/or imino acidsconnected to one another.

[0066] A polypeptide is defined as a chain of greater than 50 aminoacids and/or imino acids connected to one another. In general, peptidesand polypeptides are linked by peptide bonds. However, other bonds thatmay be substituted for peptide bonds are known in the art. Peptides andpolypeptides may be composed of naturally or non-naturally occurringamino acids or of amino or imino acid substitutes known in the art.

[0067] A protein is a large macromolecule composed of one or morepolypeptide chains.

[0068] For the purposes of this invention, the term “mucin-type protein”or “mucin-type protein that is derived from a mucin” refers to a proteinthat is greater than 50 amino acids in length and that has an amino acidsequence the same or similar to a mucin protein. Mucin-type proteins maybe isolated from a biological source, synthesized recombinantly,synthesized by chemical coupling of amino acids, peptide or polypeptideunits, or formed by combining any of these through chemical coupling.

[0069] The term “mucin-type peptide” or “mucin-type peptide that isderived from a mucin” refers to a molecule or macromolecule that isequal to or less than 50 amino acids in length and that has an aminoacid sequence the same as or similar to all or a part of a mucinprotein. Mucin-type peptides may comprise one or more tandem repeatregions. Mucin-type peptides are generally synthesized by chemicalmethods but can also be isolated from biological sources or synthesizedby recombinant methods.

[0070] The term covalent bond refers to a region of relatively highelectron density between nuclei which arises partly by shared-electronsand have average bond energies on the order of 200-400 kJ/mol.Non-covalent bonds are all other bonds not classified as covalent bondsand comprise ionic bonds, hydrogen bonds, Van der Waals interactions andhydrophobic interactions and have bond energies on the order of 2-50kJ/mol.

[0071] An epitope or single antigenic determinant is either a group ofamino acids on a protein surface or groups of sugar residues in acarbohydrate which is responsible for combining with the antibody orT-cell receptor combining site.

[0072] An amino acid sequence is similar to a reference amino acidsequence when it has at least 70% sequence identity to the referenceamino acid sequence. In a preferred embodiment, the sequence identity isat least 75%, 80%, 85% or 90%. In a more preferred embodiment, the aminoacid sequence is at least 95%, 96%, 97%, 98% or 99% identical to thereference sequence.

[0073] The term immobilized refers to a molecule that reachesequilibrium upon being absorbed or affixed to a chromatography medium,wherein no more that 2% of the total amount is washed off during eachexperimental analysis.

[0074] Compositions for Mucin Immobilized Chromatography

[0075] One embodiment of the invention is drawn to a composition thatcan be used for MIC. In one embodiment, the composition comprises aprotein non-covalently immobilized to a solid support matrix, whereinsaid protein is a mucin-type protein or mucin-type peptide and the solidsupport matrix comprises a surface with covalently attached amphiphilicmolecules.

[0076] The MIC composition comprises a solid support matrix that iscomprised of an inorganic molecule, a polymer, or a copolymer. In oneembodiment, the solid support matrix is an inorganic molecule that isselected from the group consisting of silicates, aluminas,hydroxyapatites, zeolites, germanates or phosphates. In a preferredembodiment, the inorganic is a functionalized silica gel. In a morepreferred embodiment, the functionalized silica gel is 3-aminopropylsilica, 1-allyl silica, 3-(3,4-cyclohexyldiol)propyl,3-(diethylenetriamino)propyl, 4-ethyl benzenesulfonamide,3-mercaptopropyl, propionyl chloride, 3-(2-succinic anhydride)propyl,and 3-(ureido)propyl.

[0077] In another preferred embodiment, the solid support matrix is apolymer that is agarose, dextran, polystyren, polyvinyl alcohol,polymethylacrylate, polymethylmethacrylate, acrylamides. In anotherembodiment, the solid support matrix is a copolymer is selected from thegroup consisting of polyethyleneglycol-co-polystyrene,polystyrene-co-divinylbenzene, sulfonated styrene-divinylbenzene,polyvinylchloride-co-vinylacetate, bis-acrylamide-azalactone,N-vinylpyrrolidone-co-divinylbenze, polyethylene-co-butyl methacrylate,poly(lactic-co-glycolic acid), lignin-cresol copolymers, dextran-agarosecopolymers, glycidyl methacrylate-ethylene dimethacrylate copolymers,poly(lactide-co-caprolactone) and polyacrylic copolymers.

[0078] The invention also provides an amphiphilic molecule covalentlyattached to the surface of a solid support matrix. The amphiphilicmolecule is one that can bind to the surface of the matrix and present ahydrophilic domain that extends away from the matrix surface. Withoutseeking to be bound by any theory, it is believed that the interactionof the hydrophobic domain of the amphiphilic molecule with the matrixforms a hydrophobic matrix for non-covalent entrapment of molecules,particularly mucin-type proteins and/or mucin-type peptides.

[0079] In one embodiment, the amphiphilic molecule is selected fromphospholipids, phosphoglycerides, spingolipids, prostaglandins,saturated fatty acids, unsaturated fatty acids or soaps. In anotherembodiment, the amphiphilic molecules are commercially available,membrane-forming lipids that generally contain one polar head-group withtwo non-polar alkyl sidechains and wherein at least one of thesidechains contains an ω-carboxyl functional group for covalent linkageto an amine modified silica surface (Regis Technologies, Morton Grove,Ill.). In a preferred embodiment, the commercially available amphiphilicmolecules are membrane forming ligands such as phosphatidyl choline(PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG),phosphatidic acid (PA), and phosphatidylserine (PS) (see, e.g., Pidgeonet al., Anal. Chem. 66, 2701-2709 (1994)). In a more preferredembodiment, the amphiphilic molecules are those described in U.S. Pat.Nos. 4,927,879 and 4,931,498 and references incorporated therein. In apreferred embodiment, the amphiphilic molecule is a phospholipid. Inanother preferred embodiment, the amphiphilic molecule is phosphatidylcholine.

[0080] The composition comprises a mucin-type protein, mucin-typepeptide or a combination thereof. The composition may comprise one ormore types of mucin-type protein, mucin-type peptides, or combinationsthereof. The mucin class of proteins consists of 13 proteins to date, ofwhich ten are present in the gastrointestinal tract (Cornfield et al.,Front. Biosci. 6, dl321-1357 (2001)). Mucins from different mucosalsurfaces can be either membrane-bound or secreted. Mucins commonlyfeature tandem repeats (TR) of amino acid sequences that are largelycomprised of serine, threonine, alanine, glycine, and proline (see,e.g., Table 1). MUC5AC and MUC6 are expressed largely in the epitheliaof the stomach. MUC2 is expressed in both small and large intestineportions of the gastrointestinal tract. The mucin-type proteins maycomprise all or a part of the amino acid sequence or carbohydratecomponents of a naturally-occurring mucins. In a preferred embodiment,the mucin-type protein comprises the entire amino acid sequence andcarbohydrate components of a naturally-occurring mucin. In a preferredembodiment, the mucin-type peptides generally comprise only a part of anaturally-occurring mucin. TABLE 1 Type and tandem repeat amino acidsequences in human mucins. Chromosomal Mucin Location Type* TandemRepeat MUC1 1q21 MB PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:1) MUC2 11p15.5 SPTTTPPITTTTTVTPTPTPTGTQT (SEQ ID NO:2) MUC3 7q22 MB HSTPSFTSSITTTETTS(SEQ ID NO:3) MUC4 3q29 MB TSSASTGHATPLPVTD (SEQ ID NO:4) MUC5AC 11p15.5S TTSTTSAP (SEQ ID NO:5) MUC5B 11p15.5 S SSTPGTAHTLTMLTTTATTPTATGSTATP(SEQ ID NO: 6) MUC6 11p15.5 S SPFSSTGPMTATSFQTTTTYPTPSHPQTTLPT (SEQ IDNO: 7) HVPPFSTSLVTPSTGTVITPTHAQMATSASIH STPTGTIPPPTTLKATGSTHTAPPMTPTTSGYSQAHSSTSTAAKTSTSLHSHTSSTHHPEVTPT STTTITPNPTSTGTSTPVAHTTSATSSRLPTPFTTHSPPTGS MUC7 4q13-q21 S TTAAPPTPSATTPAPPSSSAPG (SEQ ID NO: 8) MUC812q24.3 S TSCPRPLQEGTPGSRAAHALSRRGHRVHELPT (SEQ ID NO: 9) SSPGGDTGF MUC91p13 S VGHQSVTPGEKTLTS (SEQ ID NO: 10) MUC11 7q22 SSGLSEESTTSHSSPGSTHTTLSPASTTT (SEQ ID NO: 11) MUC12 7q22 MBSGLSQESTTFHSSPGSTHTTLSPASTTT (SEQ ID NO: 12)

[0081] In a preferred embodiment, the composition comprises a mucin-typeprotein or mucin-type peptide derived from or the same as MUC2, MUC5AC,MUC5B, or MUC6. In a more preferred embodiment, the compositioncomprises MUC5AC or MUC2. In a more preferred embodiment the compositioncomprises MUC5AC.

[0082] In another aspect, the composition comprises a mucin-type proteinor peptide having particular types of glycosylation. Mucin-type proteinsor peptides may be O-linked glycosylated at threonine and serineresidues found in the polypeptide chain. In one embodiment of thisinvention, the mucin-type protein or peptide contains threonine andserine amino acid residues that are fully O-linked glycosylated at allhydroxyl groups in the polypeptide, partially O-linked glycosylated, ornon-glycosylated at the hydroxyl group of the amino acid side chain. Inanother embodiment of the invention, the mucin-type protein or peptideis between 50-80% O-linked glycosylated. The polypeptide may beglycosylated with monosaccharides, disaccharides, oligosaccharides, andpolysaccharides. A preferred oligosaccharide is Gal β(1-3)-GalNAcα(1-0)-, Gal β(1-3)-[GlcNAc β(1-6)]-GalNAc α(1-0)-, GlcNAc β(1-3)-GalNAcα(1-0)-, GlcNAc β(1-3)-[GlcNAc β(1-6)]-GalNAc α-(1-0)-, GalNAcα(1-3)-GalNAc α-(1-0)-, or Gal β(1-3)-[Gal β(1-6)]GalNAc α(1-0)-. Inanother embodiment, a disaccharide is Galβ(1-3)GalNAc α-. In anotherembodiment, a monosaccharide is GalNAc.

[0083] Similarly, mucin-type proteins or peptides may be N-linkedglycosylated at asparagine amino acid residues found in the polypeptidechain. N-glycosylation may occur at asparagine residues wherein theasparagine is present in the sequence of amino acids Asn-X-Ser orAsn-X-Thr, wherein X is any amino acid other than proline and asparticacid. Thus, another embodiment of the instant invention, the mucin-typeprotein contains asparagine amino acid residues that are fully N-linkedglycosylated at all side-chain amine groups in the polypeptide,partially N-linked glycosylated, or non-glycosylated at the amide groupof the amino acid side chain. The polypeptide may be glycosylated onasparagine residues with monosaccharides, disaccharides,oligosaccharides, or polysaccharides. Preferably, the side chain isN-linked glycosylated with a hexasaccharide, GlcNAc₃Man₃.

[0084] In another embodiment, the composition comprises a mucin-typepeptide that is non-glycosylated. In another embodiment, the mucin-typepeptide, whether glycosylated or not, is an epitope associated withmucin from either malignant or normal cells. In a preferred embodiment,the epitope is an amino acid sequence. In another preferred embodiment,the epitope is a sequence of sugar residues.

[0085] This invention also provides a composition wherein the mucin-typeprotein or mucin-type peptide is a secreted form or membrane-bound formof mucin. The mucin-type protein or mucin-type peptide may be derivedfrom or is the same as a mucin derived from the GI tract, eye, trachea,lungs, salivary glands, sweat glands, breast, reproductive tract,pancreatic duct, gall bladder or urinary tract, e.g. the urethra andbladder. In a preferred embodiment, the mucin-type protein or mucin-typepeptide is derived from or is the same as a mucin derived from the GItract. In another embodiment, the mucin is derived from a normal ormalignant cell, tissue or organ.

[0086] In a further embodiment, the instant invention provides amucin-type protein that is a mammalian mucin. The mammalian mucin may bederived from any species, and includes, without limitation, human, apes,monkey, rat, pig, dog, rabbit, cat, cow, horse, mouse, rat, and goat. Inanother embodiment, the present invention relates to a mucin-typeprotein or mucin-type peptide that comprises one or more tandem repeatsequences selected from the group consisting of SEQ ID NOS:1-12.

[0087] The composition may further comprise other mucus layercomponents. Components of the mucus layer other than mucins includelipids, other non-mucin proteins, DNA, and carbohydrates. In anotherpreferred embodiment, the composition further comprises non-mucinproteins. Non-mucin proteins that may interact with mucins includelysozyme, lactoferrin, immunoglobulins, protease inhibitors, growthfactors, cytokines, defensins, β-galectins, trefoil factor peptides, IgGgamma Fc binding proteins, heparin and heparin sulfate,lipopolysaccharide binding proteins, and serum amyloid A proteins.

[0088] Examples of proteins specifically secreted in thegastrointestinal tract that interact with mucin include proteaseinhibitors such as, without limitation, 1-antitrypsin inhibitor,pancreatic secretory trypsin inhibitor (PSTI), and secretory leukocyteproteinase inhibitor; growth factors, which include mucosal integritypeptides (e.g., transforming growth factor-alpha and PSTI), luminalsurveillance peptides (e.g., epidermal growth factors (EGF)), and rapidresponse peptides (e.g., trefoil factor family (TFF) peptides). The TFFpeptides, sometimes referred to as P-domain peptides, include humanspasmolytic peptide (TFF1), TFF2 and intestinal trefoil peptide (TFF3).Other proteins, such as alpha-defensins, HD5 and HD6, beta-defensin hBD1and beta-galectins-1, 3 and 4 are also found in the gastrointestinaltract. See Cornfield et al., Front. Biosci. 6, dl321-1357 (2001).

[0089] The non-mucin proteins and mucin-type proteins and peptides maybe isolated from a biological source or prepared by recombinant DNAmethods, synthetic chemical routes, or a combination of any of thesemethods. Methods of preparing mucins from biological sources arewell-known in the art. See Larhed et al., J. Pharm. Sci. 86, 660-665(1997); Thorton et al., “Glycoprotein Methods and Protocols: The Mucins”in Methods in Molecular Biology, A. P. Corfield, Ed., Humana Press Inc.,Totowa, N.J., 125, 77-85 (2000). Methods of preparing mucin-typeproteins and mucin-type peptides recombinantly are known in the art. SeeHu et al, Cancer Research, 53, 4920-4926 (1993); Dolby et al., ProteinExpression and Purification, 15, 146-154 (1999). Preparation of peptidesand proteins by chemical synthesis is known in the art. See Solid-PhasePeptide Synthesis, G. B. Fields, Ed., 1st ed., Academic Press, New York,289 (1997); Fontenot et al., Peptide Research, 6, 330-336 (1993). In apreferred embodiment, the mucin-type protein is derived from abiological source. The mucin-type protein may be highly purified or maybe a crude, relatively heterogeneous form. In a more preferredembodiment, the mucin-type protein is derived from pig.

[0090] Recombinant DNA methods include expressing the protein or peptideof interest from a host cell that comprises a nucleic acid moleculecomprising a nucleic acid sequence encoding the protein or peptide ofinterest. The host cell may comprise a vector comprising the nucleicacid sequence. The vector may further comprise an expression controlsequence operably linked to the nucleic acid sequence. In a preferredembodiment, the nucleic acid sequence encodes an amino acid sequenceselected from the group consisting of a mucin-type protein and amucin-type peptide. The mucin-type peptide may comprise a mucin epitope.The host cell may be a prokaryotic or a eukaryotic cell. In a preferredembodiment, the prokaryotic cell is an E. coli cell. In anotherpreferred embodiment, the eukaryotic cell is selected from the groupconsisting of yeast, insect, and mammalian cells.

[0091] Methods for Preparing Chromatography Medium

[0092] Another aspect of the present invention is to provide a methodfor preparing a composition comprising a mucin-type protein or amucin-type non-covalently immobilized to a solid support matrix, whereinsaid matrix is surface modified with an amphiphilic molecule, comprisingthe step of mixing a solution comprising a mucin-type protein and/ormucin-type peptide with a solid support matrix that is surface modifiedwith an amphiphilic molecule under conditions in which the mucin-typeprotein, mucin-type peptide or combination thereof is adsorbed to thematrix. The mixture of mucin-type protein to mucin-type peptide canrange from 1:99 parts mucin-type protein:peptide to 99:1 mucin-typepeptide: protein.

[0093] In a preferred embodiment, the mucin-type protein or mucin-typepeptide is a 10% (w/v) solution of mucin-type protein or peptide in asolvent. In another preferred embodiment, the solvent is polar. In amore preferred embodiment, the polar solvent is acetone or DPBS/Brij-35.In yet another preferred embodiment, the matrix is present in a ratio of1:4 to 4:1 (w/v) to said 10% (w/v) solution of protein/peptide in asolvent. In a more preferred embodiment, the matrix is present in aratio of about 2:1 (w/v) to said 10% (w/v) solution of protein/peptidein a solvent.

[0094] In another preferred embodiment of the instant invention, themixing comprises the step of stirring the mixture in a closed vial at atime and temperature sufficient to allow the mucin-type protein ormucin-type peptide to equilibrate with the solid support matrix. In oneembodiemnt, the equilibrium takes about 1-2 hrs at a temperature up to25° C. In yet another preferred embodiment, the method comprises thesteps of washing, drying or subsequently washing and drying the mixture.In a more preferred embodiment, the drying step comprises evaporatingsaid solvent in air or by rotoevaporation. In yet another preferredembodiment, the dried mixture is packed in a column for use in liquidchromatography, high performance liquid chromatography, or fastperformance liquid chromatography; coating a glass slide for thin layerchromatography; or coating a multi-well plate for HTS.

[0095] The method may optionally comprise adsorbing other molecules fromthe mucus layer, including other proteins, DNA, RNA, lipids orcarbohydrates. Other molecules may be immobilized using methods known inthe art. See, e.g., Zhang et al. in which nicotinic acetylcholinereceptors (nAChR) were immobilized to IAM HPLC supports (Anal. Biochem.264, 22-25 (1998)) and Chui et al., in which enzymes such asα-chymotrypsin and trypsin were immobilized to IAM supports (Anal.Biochem. 201, 237-245 (1992)). Macromolecular structures such as ratliver microsomes have also been immobilized to IAM supports(Alebić-Kolbah and Wainer, J. Chromatography 646, 289-295 (1993)).

[0096] In one embodiment of the invention, a mucin-type protein is boundto a phosphatidylcholine carrier. In a preferred embodiment, porcineMUC5AC is used. In a more preferred embodiment MUC5AC is immobilized toIAM carrier particles (MUC5AC-IAM) at weight ratios of 10:1, 4:1, 2:1,1;1, 1:2, 1:4, and 1:10. In a even more preferred embodiment of theinstant invention, a 2:1 ratio of MUC5AC to IAM particles is used. Inanother embodiment, chromatography columns are packed with immobilizedMUC5AC-IAM particles having ratios of 1:4, 1:2, 1:1, and 2:1 (MUC5AC toIAM).

[0097] Methods for Determining in vitro Drug Permeability Using MICMedia

[0098] For oral drug administration, bioavailability can be calculatedby the following:

F=fa(1−E _(G))(1−E _(H)),  [1]

[0099] where F is bioavailability of a compound, fa is the extent ofabsorption in intestinal mucosa, E_(G) is cytosolic localized metabolismin the enterocyte and E_(H) is the extraction in the liver includingboth metabolism and biliary secretion (Lennernäs, H., “Human PerfusionStudies” in Oral Drug Absorption: Prediction and Assessment, Dressman,J. and Lennernäs, H., Eds., 106, 99-117 (2000)). The effectivepermeability of a drug may be used to estimate fa. Thus, determining theeffective permeability is an important part of estimatingbioavailability.

[0100] Effective permeability is estimated from the thickness of themucin-type protein or mucin-type peptide layer non-covalentlyimmobilized to the solid support matrix, the membrane diffusioncoefficient of the drug, and the membrane equilibrium coefficient forthe mucin-type protein or mucin-type peptide layer non-covalentlyimmobilized to the solid support matrix. The membrane equilibriumcoefficient is determined from the volume of the mobile phase, theinterstitial volume of the stationary phase, and the partition ratio,which is further determined from the retention time of the drugcandidate and the void volume of the column, as discussed below.

[0101] The effective permeation of a drug through a membrane is given bythe equation, $\begin{matrix}{{P_{eff} = \frac{D_{m}K_{m}}{L}},} & \lbrack 2\rbrack\end{matrix}$

[0102] where D_(m) is the membrane diffusion coefficient of the solute,K_(m) is the membrane equilibrium constant, and L is the membranethickness (Stein, W., Transport and Diffusion Across Cell Membranes,Academic Press, Orlando, Fla. (1986)). D_(m) depends on the molecularsize of the drug and is proportional to the inverse of molar volume ofthe drug (Pidgeon and Ong, Chemtech. June, 38-48 (1995); Xian andAnderson, Biophys. J., 66, 561-573 (1994)). Since molecular weight, MW,is proportional to molecular size, D_(m) may also written as,$\begin{matrix}{D_{m} = {\frac{1}{MW}.}} & \lbrack 3\rbrack\end{matrix}$

[0103] Thus, Dm can be calculated from the molecular weight.Furthermore, K_(m) is calculated by the following equation,

K _(m)=(V _(m) /V _(s))k′,  [4]

[0104] where V_(m) is volume of mobile phase, V_(s) is the internalvolume of the stationary phase, and k′ is the partition ratio (Pidgeonand Ong, Chemtech. June, 38-48 (1995)). The partition ratio isdetermined by the equation,

k′=(t _(r) −t _(o))/t _(o),   [5]

[0105] where t_(r) is the retention time of the drug and t_(o) is thevoid volume of the column. Both t_(r) and t_(o) can be experimentallydetermined in order to obtain k′.

[0106] The thickness of the mucin layer, L, may be determined by anymethod know in the art. In a preferred embodiment, the mucin-layerthickness is determined by confocal microscopy. According to thisinvention, all values of L measured by this method are determined byaveraging the thickness of the mucin layer on each of 10-15 beads. Theuse of the term “about” in connection with defining thickness (L) refersto a value that is similar to the defined thickness. For example, an Lvalue of “about” a particular defined thickness would be a thicknesswithin 10%, preferably 5%, more preferably 2%, of the defined thickness.Furthermore, detection of the layer thickness by confocal microscopy canbe accomplished using a mucin-specific antibody. In a more preferredembodiment, the antibody may be an anti-MUC5AC antibody labeled withbiotin and the streptavidin labeled with Alexa Fluor 488.

[0107] Equation 2 can be used to determine K_(m) for a given MIC medium.Both D_(m) and L are known from the discussion above, and P_(eff) can becalculated for various drugs having known in vivo absorption. Forexample, a number of drug permeability coefficients, P_(eff), derivedfrom in vivo experiments are listed in literature. Plotting variousknown drug permeability coefficients as a function of the measured k′values for the same drugs on a separation media will result in acorrelation equation that can be used to determine other P_(eff) valuesnot found in literature. See Example 5 below. Once K_(m) has beencalculated in this manner for a specific MIC medium, compounds havingunknown P_(eff) can be determined.

[0108] Methods for Estimating Drug Permeability

[0109] The MIC media may be used in any method, including, inter alia,batch chromatography or column chromatography, which includes liquidchromatography, high performance liquid chromatography, and fastperformance liquid chromatography. The MIC media may also be used tocoat a slide or plate for thin layer chromatography. In anotherembodiment, the MIC media may be used to coat a multi-well plate for HTSor other high-throughput analyses. The MIC media may be used to separatecomponents in a mixture from one another using methods well-known in theart.

[0110] Another aspect of the present invention is to provide in vitromethods for estimating drug absorption through the mucus layer. Thismethod comprises determining an effective permeability coefficient(P_(eff)) for a drug compound using MIC media.

[0111] In one aspect, the invention provides a method of estimating thepermeability coefficient of one or more drugs by contacting the drugwith the MIC media and measuring the degree of binding of the drug onthe MIC media and estimate the drug permeability coefficient. In apreferred embodiment, the contacting step is performed by loading thedrug on a column comprising the MIC media and the measuring step isperformed by measuring the retention time of the drug on the column. Inone embodiment, the drug permeability coefficient is estimated for asingle drug.

[0112] In another preferred embodiment, permeability coefficients of anumber of drugs are measured either sequentially, by contacting eachdrug with the MIC media one by one and measuring binding of the drug tothe media, or simultaneously, by contacting a mixture of more than onedrug with the MIC media and measuring binding of the drug to the media.In another embodiment, the MIC media comprises types and/or amounts ofmucin-type protein or mucin-type peptide to mimic the mucus layer in aspecific cell, tissue or organ. These include the mucus layer of the GItract, eye, trachea, lungs, salivary glands, sweat glands, breast,reproductive tract, pancreatic duct, gall bladder or urinary tract.

[0113] In another aspect, the method may be used to compare the relativepermeability of one or more drugs to each other or to drugs having aknown permeability. The method comprises contacting the MIC media with adrug of interest and measuring the degree of binding of the drug to theMIC media, then comparing the degree of binding to that measured for oneor more other drugs. The other drug may be one having an unknown drugpermeability coefficient or may be one having a known drug permeabilitycoefficient. The method may be used to determine whether the drug ofinterest has a greater or lesser relative permeability compared to theother drug. Further, by comparing the drug of interest to one or moreother drugs having a known permeability coefficient, one may estimatethe permeability coefficient for the drug of interest. The degree ofbinding may be measured sequentially or simultaneously. The degree ofbinding may be determined using column chromatography or by batchchromatography. See Example 5.

[0114] Another embodiment of the invention is to provide a method forestimating effective permeability coefficients of one or more drugs intwo or more physiological states by changing the amount and/or type ofmucin-type protein or mucin-type peptide in the MIC media. In onepreferred embodiment, the MIC media comprises types and/or amounts ofmucin-type proteins or mucin-type peptides that mimic the mucus layer ina healthy state or in a diseased states. In another embodiment, themethod provides a method for estimating effective permeabilitycoefficients of one or more drugs in two or more physiological states bychanging the mobile phase of the media. In a preferred embodiment, thepH, osmolarity and/or surfactant concentration or type is altered tomimic a particular physiological state.

[0115] The MIC media may be used to mimic the healthy or diseased stateof any mucosal layer, including the gastrointestinal tract, reproductivetract, urinary tract, eye, mouth, salivary glands, pancreatic glands,sweat glands or gall bladder. In a preferred embodiment, the diseasedstate is epithelial cell cancer. The types and amounts of mucin-typeproteins and mucin-type peptides that are representative of healthystates and disease states of the intestine can be determined by onehaving ordinary skill in art (Cornfield et al., Frontiers in Bioscience,6, d1321-1357 (2001); Bhavanadan, V. P., Glycobiology, 1, 493-503(1991); Zotter et al., Cancer Rev., 11-12, 55-101, (1988); Fink, M. P.,Crit. Care Med., 19(5), 627-641 (1991)).

[0116] In another embodiment, the different physiological states aredifferent developmental stages, e.g., infant, child, adolescent andadult. In another embodiment, the different physiological states may bemucosal layers from different cells, tissues, or organs. For example,one may compare estimated drug absorption in the mouth, gastrointestinaltract and reproductive tract to determine the best method ofadministration.

[0117] A further embodiment of this invention is to provide a method foremulating an absorption process in a system comprising more than oneepithelial mucosal layer. The method comprises estimating drugpermeability coefficients using a series of chromatography columns eachhaving a different amount and/or type of a mucin-type protein,mucin-type peptide, or combination thereof immobilized to achromatography medium, wherein the different amounts and/or types ofmucin-type proteins or mucin-type peptides mimic the different mucuslinings in the system. In a preferred embodiment, the mobile phase maybe altered to mimic the different extracellular fluids in the system. Ina preferred embodiment, the system is the digestive system. For example,the thickness of the mucus lining along the gastrointestinal tractvaries according to the cellular and glandular tissues associated withlocation. The thickness of the mucus layer in the esophagus has beenmeasured to be between 83-107 μm, the stomach between 92-196 μm, theduodenum between 11-21 μm, the proximal colon between 59-155 μm, thesigmoid colon between 19-113 μm, the distal colon between 66-202 μm, andthe rectum between 101-209 μm (see, e.g., Corfield et al., Frontiers inBioscience 6, 1321-1357 (2001) and references therein).

[0118] In a preferred embodiment, the invention provides columnchromatography where the stationary phase comprises MUC5AC-IAM and themobile phase comprises an aqueous buffer solution employed at conditionsmatching those of the GI tract. For instance, to simulategastrointestinal conditions, sample sets may be analyzed using a mobilephase of between pH 2 and pH 8 using MIC media. Other variables such asosmolarity, surfactant concentration, lipid concentration, digestiveenzyme concentration, bile acid concentration, and other GI componentsmay also be added to the mobile phase to match conditions representativeof the GI tract.

[0119] Another embodiment of the instant invention is a kit comprisingthe MIC media. The kits may be used to estimate absorption of one ormore drugs. In one embodiment, diagnostic kits can include but are notlimited to one or more standards, such as a drug having a known in vivodrug permeability coefficient and one or more MIC media.

[0120] Another embodiment of the instant invention comprises a methodfor using MIC in HTS. In a preferred embodiment, HTS includes HPLC orELISA.

EXAMPLES

[0121] The following materials were used in the examples set forthbelow.

[0122] Materials

[0123] Type II mucin, crude from porcine stomach, was obtained fromSigma Chemicals (St. Louis, Mo.), polyethylene glycol dodecyl ether(Brij-35 P) was purchased from Fluka Chimica, benzyl alcohol waspurchased from Fisher (Pittsburgh, Pa.), acetone was obtained fromFisher, and Dulbecco's phosphate buffered saline (DPBS) was obtainedfrom JRH Biosciences (Lenexa, Kans.). Model drug compounds differing intheir physiochemical properties and listed in Table 3 were purchasedfrom Sigma Chemicals (St. Louis, Mo.). Mucin (MUC-5AC) antibody wasobtained from NeoMarkers, Lab Vision Corporation (Fremont, Calif.) andbiotin-XX goat anti-mouse IgG (H+L) and streptavidin linked to AlexaFluor 488 were purchased from Molecular Probes (Eugene, Oreg.).Immobilized artificial membrane columns and loose packing materialcontaining silica modified with diacylated phosphatidyl choline ligandsand endcapped with C10/C3 alkyl chains (IAM.PC.DD2 and loose IAM.PC.DD2,10×4.6 mm, 12 μm, 300 Å) were purchased from Regis Technologies (MortonGrove, Ill.). Dialysis tubing having a molecular weight cutoff (MWCO) of8,000 was purchased from Spectrum Laboratories (Houston, Tex.). BCAProtein assay kits were obtained from Pierce Endogen (Rockford, Ill.).

Example 1

[0124] Non-Covalent Immobilization of Mucin to IAM PhosphatidylcholineMaterial

[0125] In order to investigate the adsorption of mucin to the columnpacking material, the ratio of mucin to IAM.PC.DD2 was varied. Aliquotsof 20 mg of IAM.PC.DD2 were with varying amounts of 10% (w/v) mucin ineither acetone or mobile phase solution (MPS) [DPBS (0.05M), Brij-35(0.02 M), and benzyl alcohol (1% v/v) at pH 7 or pH 3] to give ratios of10:1, 4:1, 2:1, 1:1, 1:2, 1:4, and 1:10 (w/w) of IAM.PC.DD2 to mucin.Each mixture was incubated at room temperature (rt) for 1 hour (h) withconstant mixing prior to being washed 5 times with MPS. After each wash,the samples were sedimented by centrifugation (at 3000 g for 10 sec) andthe supernatants were monitored for protein content by BCA assayaccording to manufacturer's instructions. Solutions containing a knownquantity of mucin were used to generate standard curves for the BCAassays. The amount of the mucin absorbed to IAM.PC.DD2 was quantified.Without being bound by theory, applicants believe that the nature of thenon-covalent interaction between mucin and IAM.PC.DD2 is electrostatic.

[0126] Large scale preparation of packing material for MIC columns wasperformed by mixing 300 mg of IAM.PC.DD2 with 10% (w/v) mucin in acetoneto yield 4:1, 2:1, 1:1, 1:2, and 1:4 (w/w) ratio of IAM.PC.DD2 to mucin.The mixtures were stirred in closed vials for 1 h at room temperaturebefore being dried. Four columns (10×4.6 mm) having mucin to IAM-PCratios of 1:4, 1:2, 1:1, 2:1 were prepared by Regis Technologies usingthis material. Table 2 and FIG. 1 illustrate an optimization plotbetween the mount of mucin absorbed to IAM beads versus the weight ratioof mucin to IAM beads. TABLE 2 Binding optimization results betweenmucin and IAM beads. Initial Ratio Mucin Absorbed to Final Ratio(mucin:IAM) IAM Beads (mg) (mucin:IAM) 10:1  6.51 1.34 4:1 7.40 1.46 2:17.22 1.46 1:1 2.50 0.47 1:2 1.21 0.24 1:4 0.81 0.16  1:10 0.05 0.01

Example 2

[0127] Analysis of Immobilized Mucin on Phosphatidylcholine Carrier (PC)with Transmission Electron Microscopy (TEM)

[0128] PC carriers with and without immobilized mucin were fixed with aruthenium-osmium mixture using the modified protocol of Luft (Anal. Rec.171, 369-416 (1977)). Carriers were then sectioned to a thickness of120-200 nm. Sample sections were contrasted with uranyl acetate and leadcitrate and were fixed on carbon-stabilized, formvar-coated, 50 mesh,copper grids. A Hitachi 7110 Scanning Transmission Electron Microscopewas used to acquire images.

[0129] FIGS. 2(a) and 2(b) illustrate the immobilization of MUC-5AC tothe IAM particles as confirmed by TEM analyses. The Luft'sfixation-staining technique (Luft, Anal. Rec. 171, 369-416 (1977))provided low electron contrast for visualization of mucopolysaccharides.The images revealed that MIC particle surfaces appear to have a hazy,ill-defined edge which was opposite of the sharp edge found innon-immobilized IAM beads (FIG. 2(c)). The observed electron densesurface coatings ranged from 14 nm to 38 nm for the MUC5AC-IAMparticles.

Example 3

[0130] Detection of Mucin Membrane Thickness with Confocal Microscopy

[0131] Mouse monoclonal antibodies (1 μg/ml) having cross-reactivity toMUC5AC were immobilized to surfaces of MIC beads having ratios of 0:1,1:4, 1:1 and 4:1 mucin to IAM beads by incubation at rt for 1 hr withconstant vortexing. The beads were washed five times with DPBS. Biotinlabeled goat anti-mouse antibodies (1 μg/ml) were then reacted withthese beads at rt for 1 h with constant mixing. The beads were thenwashed five times with DPBS. Streptavidin labeled with Alexa Fluor 488(5 μg/ml) was then added to the coated beads. The beads were washedbefore being analyzed by confocal microscopy using a Biorad Radiance2000 MP confocal imaging system attached to an Olympus BX50WI uprightmicroscope. The thickness of the absorptive layer was calculated usingan intensitometric approach (Leung and Jeun, Microscopical Society ofCanada Bulletin, 20, 26-33 (1992)).

[0132] Mucin thickness was determined by averaging 10-15 measuredthicknesses for each ratio of mucin to IAM beads used. FIG. 3 shows across-section of MUC5AC-IAM beads where the mucin layer is visualized bystaining as indicated by the rings present. Due to the variability insectioning, some of the beads appeared to have thicker rings thanothers. In a preferred embodiment, confocal measurements for a series ofratios of mucin to loose PC particles (1:4, 1:1, and 4:1) weredetermined. The thickness increased from 0.3 μm, the thickness of a barephospholipid layer on a silicon carrier, to 0.38 μm, the thicknesscorresponding to the 4:1 ratio of mucin to IAM particles beads. Withoutbeing bound by theory, applicants believe that the mucin partiallyintercalates the phospholipid layer. Additionally, the thickness ofmucin:IAM beads having ratios of 1:4 and 1:1 were 0.326 and 0.35 μm,respectively. A plot of mucin:IAM ratio to measured thickness wasapproximately linear as shown in FIG. 4 and could be used to extrapolateother mucin:IAM bead ratios such as 2:1 (0.36 μm). Membrane thickness,L, was then used to calculate K_(m) as discussed above.

Example 4

[0133] Determination of Mucin and Drug Interaction

[0134] A known amount of a drug compound or an enzyme was dissolved in10% (w/v) mucin in mobile phase solution. The sample was incubated for 1h with constant mixing at rt before being transferred to dialysis tubinghaving a MWCO of 8000. The sample was then dialyzed against deionized(DI) water overnight at rt before further dialysis with fresh DI waterfor another 12 h at rt. The dialyzed media were analyzed by reversedphase HPLC to determine the amount of drug released to the environment.The percent of drug immobilized to mucin was determined by subtractingthe amount of drug released into dialysis medium from the amount of drugmixed with the mucin.

Example 5

[0135] Estimation of Drug Absorption by High Performance LiquidChromatography (HPLC)

[0136] Columns containing 1:4, 1:2, 1:1, and 2:1 wt % of mucin to PC IAMparticles were packed and used to determine drug retention times. It wasobserved that the column containing the 1:4 ratio provided the longestdrug retention times. Without being bound by theory, applicants believethat these results are related to the physiochemical properties,specifically hydrophobicity, of mucin itself. For example, theimmobilization of mucin most likely involves the non-covalent entrapmentof hydrophobic groups from the mucin molecule with the hydrophobicpockets of the IAM molecules attached to silica. With an increase ofmucin to IAM beads, the chromatography medium becomes more hydrophobicbecause there are more hydrophobic groups from mucin available tointeract with a limited number of hydrophobic groups within the IAMbeads. The smaller 1:4 ratio of mucin to IAM beads results in a similarnumber of hydrophobic interactions between mucin and IAM. This wouldthen allow the hydrophilic groups of the mucin to be available forbinding drug candidates and would result in increased retention times ofhydrophilic drugs on the mucin to IAM chromatography medium. Forsubsequent experiments, a 2:1 column was used to determine drugretention times and calculate k′_(MIC) (see equations 1-5 and Table 4below).

[0137] The bioavailability of twenty-nine different compounds were usedfor comparing in vitro and in vivo models (see Table 3). The in vivobioavailability values for single-pass perfusion permeabilitycoefficient (Loc-I-Gut) and in vitro parameters for IAM and Caco-2models were obtained from the literature. See Table 3. TABLE 3 KnownBioavailability Values for Pharmaceutical Compounds. Bioavailability N(% Absorbed) Compound Reference 1 80 acetaminophen NA 2 68 acetylsalicylic 1 acid 2 100 acetyl salicylic 2 acid 3 20 Acyclovir 2 3 30Acyclovir 1 4 0 α-methyldopa 3 5 93 Alprenolol 2 6 100 Antipyrine 3, 4 750 Atenolol 3, 4 8 100 Caffeine 5 9 N/A carbamazepine NA 10 62Cimetidine 1 10 95 Cimetidine 2 11 100 corticosterone 2 12 0 Creatine 3,6 13 60 enalapril maleate 3, 6 13 100 enalapril maleate 7 14 60Furosemide 3, 4 15 100 Ibuprofen 1 16 100 Ketoprofen 4 17 90 Labetalol 218 16 Mannitol 2 18 17 Mannitol 8 19 95 Metoprolol 2 20 100 Naproxen 4,8 21 10 PEG 900 1, 8 22 0 PEG 4000 8, 9 22 10 PEG 4000 1 23 90Propranolol 2 23 100 Propranolol 3, 6 24 80 quinidine, HCl 1 25 13sulfasalazine 2 26 60 Terbutaline 2, 4, 7 26 73 Terbutaline 2, 4, 7 2772 Timolol 2 28 60 Verapamil 3 29 98 Warfarin 2

[0138] Chromatograms were acquired on a Thermo Separations HPLCinstrument (San Jose, Calif.) equipped with 10×4.6 mm IAM.PC.DD2,Discovery C18 column, or MIC columns. Samples were dissolved in mobilephase solution and filtered (filter pore size, 0.45 μm) prior toinjection. Elution profiles were monitored in the UV-Vis range using anisocratic method (0.3 ml/min at either 37.4° C. or 30° C. using MPS atpH 7.4). The 29 samples provided in Table 3 were loaded on the columnand their retention times were measured. From this measurement and ameasurement of the void volume (t₀), the k′_(MIC) could be determined.In addition, from the molecular weight of the compounds, D_(m) could bedetermined. P_(eff) was known for the compounds listed in Table 5. Thesecompounds, with the exceptions noted below, were used to generate thecolumn constant (V_(m)/V_(s)). See FIG. 5.

[0139]FIG. 5 shows the correlation between effective permeability,P_(eff Loc-I-Gut), and capacity factor, k′_(MIC). Since MIC is valid forpassive diffusion transport, any data corresponding to carrier mediatedtransport, such as α-methyldopa and creatine, were not included in theplot. In addition, enalapril maleate was reported to have a percentabsorption value of either 60 or 100 (Winiwarter et al., J. Med. Chem.41, 4939-4949 (1998); Lennernäs et al., Pharm. Res. 14 (5), 667-671(1997); Fagerholm et al., J. of Drug Targeting 3, 191-200 (1995)).During the HPLC analyses, it appears that enalapril maleate washydrolyzed to enalaprilat, as confirmed by the presence of two peakscorresponding to the hydrolyzed and non-hydrolyzed forms (http://www.rxlist.com/cgi/generic/enalap.htm). The value corresponding to thehydrolyzed form was used to generate FIG. 5. In addition, valuescorresponding to P_(eff Loc-I-Gut) and k′_(MIC) for both ketoprofen andnaproxen were excluded. The correlation between mean data ofP_(eff Loc-I-Gut) and K′_(MIC) was best fit to an exponential curve,y=0.0081e^(0.00634x). However, the linear fit, y=0.1115x−3.6151(R²=0.8167), was subsequently used to calculate the values ofP_(eff MIC). See Table 4. TABLE 4 HPLC Drug Permeability Values forPharmaceutical Compounds Using MIC Media. Retention Molecular K_(m)Compound Time k′_(MIC) Weight D_(m) (×10⁻⁶) P_(eff MIC) acetaminophen2.8 38.2857 151 0.0066 0.3559 0.6538 Acetyl 2.8 38.5714 180 0.00550.4477 0.6856 salicylic acid Acyclovir 2.4 33.5714 225 0.0044 0.10390.1281 α-methyldopa 2.4 33.2857 211 0.0047 0.0319 0.0963 Alprenolol 3.346.1429 286 0.0035 1.5740 1.5298 Antipyrine 5.5 77.5714 188 0.00533.4107 5.0341 Atenolol 2.2 30.4286 266 0.0038 −0.2131* −0.2223* Caffeine2.7 37.5714 194 0.0051 0.4014 0.5741 carbamazepine 4.2 59.0000 2360.0042 2.5209 2.9634 cimetidine¹ 2.4 33.2857 252 0.0039 0.0874 0.0963cimetidine² 8.5 120.429 252 0.0040 8.9021 9.8127 corticosterone 4.360.4286 347 0.0029 3.8952 3.1227 Creatine 2.2 30.4286 113 0.0088−0.0905* −0.2223* Enalapril 7.8 110.429 493 0.0020 15.4210 8.6977maleate³ enalapril 2.6 35.8571 493 0.0020 0.6790 0.3830 maleate⁴furosemide 3.5 49.0000 331 0.0030 2.2006 1.8484 ibuprofen 3.8 53.2857228 0.0044 1.9119 2.3263 ketoprofen 3.8 52.5714 254 0.0039 2.0567 2.2466labetalol 3.8 53.2857 365 0.0027 3.0559 2.3263 mannitol 2.4 33.4286 1820.0055 0.0736 0.1122 metoprolol 3.1 43.2857 685 0.0015 2.9861 1.2113naproxen 3.6 50.4286 230 0.0043 1.6642 2.0077 PEG 900 2.4 33.2857 9000.0011 0.3119 0.0963 PEG 4000 2.4 33.2857 4000  0.0003 1.3861 0.0963propranolol 5.0 70.4286 296 0.0034 4.5157 4.2377 quinidine, HCl 5.070.4286 379 0.0026 5.7804 4.2377 sulfasalazine 5.1 71.8571 398 0.00256.3063 4.3970 terbutaline 3.1 43.2857 274 0.0036 1.1961 1.2113 timolol2.6 36.1429 433 0.0023 0.6459 0.4148 verapamil 5.1 71.8571 491 0.00207.7737 4.3970 warfarin 3.9 54.7143 308 0.0032 2.7587 2.4855

[0140] TABLE 5 Effective permeability and capacity factors forpharmaceutical compounds passively absorbed. % P_(eff Loc-I-Gut)P_(eff Loc-I-Gut) P_(eff MIC) P_(eff Caco-2) Absorbed Drug (×10⁻⁴ cm/s)*σ K′_(MIC) (×10⁻⁴ cm/s)** (×10⁻⁶ cm/s)*** 100  antipyrine 5.6 1.6 77.575.03 NA 100, 90  propranolol 2.9 2.2 70.43 4.24 21.8 100  naproxen 8.04.2 50.43 2.01 74.2 100  ketoprofen 8.5 3.9 52.57 2.25 NA 95 metoprolol1.5 0.9 43.29 1.21 23.7 60 terbutaline 0.3 0.3 43.29 1.21 0.47 60furosemide 0.3 0.3 49.00 1.85 NA 60 verapamil 5.0 2.5 71.86 4.40 NA 60enalapril 0.2 0.3 35.86 0.38 NA maleate 50 atenolol 0.15 2.0 30.43 −0.220.53  0 PEG 4000 0 0 33.29 0.10 0.97  0 α-methyldopa 0.2 0.06 33.29 0.10NA  0 creatine 0.3 0.2 30.43 −0.22 NA carbamazepine 4.3 2.7 59.00 2.9619.9

[0141]FIG. 6 shows the correlation between the measured partitioncoefficient (k′) determined by MIC and the membrane equilibrium constant(K_(m)) derived from equation 2 and shown in Table 4.

[0142] The comparison of P_(eff) values as determined by MIC, Loc-I-Gut,and Caco-2 methods to in vivo percent absorption is illustrated in Table6 and FIG. 7. The data corresponds to atenolol, carbamazepine,metoprolol, naproxen, PEG 4000, and propranolol compounds.

[0143] Additionally, capacity factors for k′_(MIC) and k′_(IAM) werealso compared to in vivo percent absorption in FIG. 8. TABLE 6 In vivoand in vitro absorption values for Loc-I-Gut, MIC, and Caco-2 methods. %P_(eff Loc-I-Gut) P_(eff Loc-I-Gut) P_(eff MIC) P_(eff Caco-2) AbsorbedDrug (×10⁻⁴ cm/s)* σ K′_(MIC) (×10⁻⁴ cm/s)** (×10⁻⁶ cm/s)*** 100propranolol 2.9 2.2 70.43 4.24 21.8 100 Naproxen 8.0 4.2 50.43 2.01 74.295 metoprolol 1.5 0.9 43.29 1.21 23.7 60 terbutaline 0.3 0.3 43.29 1.210.47 carbamazepine 4.3 2.7 59.00 2.96 19.9 50 atenolol 0.15 2.0 30.43−0.22 0.53 0 PEG 4000 0 0 33.29 0.09 0.97

[0144] The P_(eff) data from the Loc-I-gut, Caco-2 and MIC methods werecorrelated with in vivo drug absorption. See FIG. 7. In the case of theCaco-2 model, the pattern of correlation was not as clear as that of IAMand MIC. The correlation between in vivo drug absorption with thepartition ratios for IAM and MIC is shown in FIG. 8 and Table 7.Similarly, the partition ratios for IAM do not correlate as stronglywith in vivo drug absorption as MIC. Thus, MIC appears to be a better invitro model for drug absorption than either the IAM or Caco-2 models.TABLE 7 Correlation between capacity factors for pharmaceuticalcompounds passively absorbed. % Absorbed Drug log k′_(MIC) log k′_(IAM)δ² 100 Antipyrine 1.89 −0.02 1.91 100 corticosterone 1.78 1.60 0.18 100Propranolol 1.85 1.75 0.10 98 Warfarin 1.74 1.15 0.59 95 Metoprolol 1.640.50 1.14 60 Terbutaline 1.64 0.50 1.14

Example 6

[0145] pH Effects on Mucin Immobilized Chromatography Retention Times

[0146] One embodiment of this invention is to simulate the healthy anddiseased states of a mucosal layer, including that of the GI tract. Forexample, a diseased state of the mucosal layer can differ in pH and/orthickness. Another embodiment of this invention is to mimic differentparts of the GI tract by changing pH. For these reasons, the influenceof pH on drug retention times was measured on columns having twodifferent mucin layer thicknesses, 0.32 μm (column prepared with a 1:2wt % of mucin to PC IAM particles) and 0.36 μm (column prepared with a2:1 wt % of mucin to PC IAM particles)(Table 8). Mucin thickness wascalculated as described in Example 3. The influence of pH on drugretention time was measured by using different mobile phase buffersadjusted to pH 3, 5 or 7 on either the 0.32 μm or 0.36 μm column. Thedata in Table 8 demonstrates that, with few exceptions, the drugretention times, calculated as partition ratios (k), at pH 3, 5 or 7were longer on the 0.32 μm column (1:2 wt % ratio of mucin to PC IAMparticles) than on the 0.36 μm column (2:1 wt % ratio of mucin to PC IAMparticles). This demonstrates that different thicknesses of mucininfluence the partition ratio and thus can be used to mimic differentmucosal linings or diseased states.

[0147] In addition, most of the drugs exhibited different retentiontimes for runs performed at pH 3, 5 or 7 on a single column. Becausemucin is comprised of hydrophilic groups that are capable of beingionized in acidic or basic environments, the corresponding interactionbetween the hydrophilic groups of mucin and those of the drug compoundwould also be modulated by change in pH environment. See Table 8. Thesedata demonstrate that MIC can be used to mimic mucosal linings ofdifferent pH and thickness. TABLE 8 pH Dependence of Partition Ratio forDrug Candidates on MIC. (1:2) (1:2) (1:2) (2:1) (2:1) (2:1) k′ k′ k′ k′k′ k′ Compound (pH 7) (pH 5) (pH 3) (pH 7) (pH 5) (pH 3) antipyrine50.67 54.00 54.00 40.67 39.00 40.43 glybenclamide 30.67 30.67 none 34.0019.00 none verapamil 55.67 30.67 27.33 45.67 24.71 34.71 nifedipine19.00 22.33 17.33 17.33 14.71 14.71 glipzide 19.00 30.67 32.33 19.0023.29 24.71 metoclorpramide 107.33 65.67 57.33 94.00 53.29 47.57di-propranol 99.00 92.33 75.67 99.00 59.00 49.00 hydrochlorothiazide67.33 70.67 70.67 55.67 13.29 14.71 phenoxybenzamine HCl 29.00 29.0020.67 15.67 17.57 20.43 ranitidine HCl 32.33 35.67 35.67 37.33 27.5727.57 trimethoprim 70.67 67.33 57.33 57.33 31.86 26.14 haloperidol110.67 44.00 30.67 95.67 34.71 23.29 creatine 39.00 39.00 39.00 32.3323.29 20.43 alprenolol 42.33 64.00 54.00 82.33 49.00 43.29acetominophenol 64.00 65.67 64.00 62.33 51.86 50.43 caffein 45.67 44.0044.00 39.00 34.71 34.71 leucine none none none none none none enalaprilmaleate 34.00 37.33 27.33 20.67 21.86 31.86 furoseimide 35.67 37.3340.67 25.67 27.57 30.43 atenolol 39.00 39.00 39.00 37.33 31.86 29.00naproxen 90.67 40.67 32.33 77.33 31.86 20.43 terbutalin 39.00 49.0045.67 35.67 37.57 34.71 ketoproten 82.33 29.00 25.67 70.67 27.57 20.433,4- 25.67 25.67 25.67 22.33 20.43 19.00 dihydrophenylalanine metoprolol47.33 50.67 50.67 35.67 41.86 36.14 corticosterone 24.00 22.33 24.0024.00 20.43 20.43 timolol 32.33 30.67 44.00 35.67 27.57 33.29sulfasalazine 24.00 85.67 87.33 22.33 67.57 70.43 labetalol 32.33 24.0024.00 132.33 90.43 79.00 warfarin 39.00 30.67 25.67 19.00 24.71 21.86acycloguanosine 25.67 15.67 25.67 24.00 20.43 20.43 cimetidine 44.0029.00 25.67 35.67 23.29 17.57 glycine none none none none none nonequinidine 99.00 87.33 49.00 132.33 63.29 37.57 amethopterin 12.33 29.0010.67 57.33 13.29 none acetylsalicyclic acid 22.33 40.67 34.00 20.6724.71 30.43

Example 7

[0148] Correlation Between Effective Permeability and Capacity Factorsfor Different Mucin Layer Thickness

[0149]FIG. 9 and Table 9 show the correlation between effectivepermeability, P_(eff Loc-I-Gut), and capacity factors k′_(MIC(2:1)) andk′_(MIC(1:2)) measured at pH 7 (see Table 8). Capacity factors werecalculated from drug retention times collected on columns having twodifferent mucin layer thicknesses, 0.32 μm (column prepared with a 1:2wt % of mucin to PC IAM particles) and 0.36 pm (column prepared with a2:1 wt % of mucin to PC IAM particles). See Example 6. A linear fit wasused to correlate mean data of P_(eff Loc-I-Gut) and k′_(MIC(2:1)) andk′_(MIC(1:2)) (R²=0.836 and R²=0.863, respectively). These data indicatea good correlation between drug permeability coefficients derived fromin vivo absorption experiments and drug partition coefficientscalculated from in vitro experiments according to this invention. MICthus serves as a valid model for estimating in vitro drug permeabilitycoefficients. TABLE 9 Effective permeability and capacity factorsk′_(MIC(2:1)) and k′_(MIC(1:2)). P_(eff Loc-I-Gut) (×10⁻⁴ k′_(MIC)k′_(MIC) Drug cm/s) * (2:1) (1:2) antipyrine 5.6 40.7 50.7 atenolol 0.237 39 furosemide 0.3 25.7 35.7 ketoprofen 8.5 70.7 82.3 metoprolol 1.535.7 47.3 naproxen 8.0 77.3 90.7 terbutaline 0.3 35.7 39 verapamil 5.045.7 55.7 enalapril 0.2 20.7 34 maleate propranolol 2.9 creatine 0.3 2425.7 PEG 4000 0 22 22

We claim:
 1. A composition comprising a mucin-type proteinnon-covalently immobilized to a solid support matrix, wherein said solidsupport matrix is surface modified with an amphiphilic molecule.
 2. Thecomposition according to claim 1, further comprising a mucin-typepeptide, which is non-covalently immobilized to said matrix.
 3. Acomposition comprising a mucin-type peptide non-covalently immobilizedto a solid support matrix, wherein said matrix is surface modified withan amphiphilic molecule.
 4. The composition according to any one ofclaims 1 or 2, wherein there is more than one species of mucin-typeprotein or mucin-type peptide immobilized to the matrix.
 5. Thecomposition according to any one of claims 1, 2, or 3, furthercomprising one or more components of a mucus layer selected from thegroup consisting of lipids, non-mucin proteins, DNA, RNA andcarbohydrates.
 6. The composition according to claim 1, wherein saidmucin-type protein is a secreted mucin, a membrane-bound mucin, or acombination thereof.
 7. The composition according to claim 3, whereinsaid mucin-type peptide is derived from a secreted mucin, amembrane-bound mucin or a combination thereof.
 8. The compositionaccording to claim 1, wherein said mucin-type protein contains threonineand serine amino acid residues that are fully O-linked glycosylated,partially O-linked glycosylated, or non-glycosylated at the hydroxylgroup of the amino acid side chain.
 9. The composition according toclaim 8, wherein said side chain is O-linked glycosylated with acarbohydrate selected from the group consisting of monosaccharides,disaccharides, oligosaccharides, and polysaccharides.
 10. Thecomposition according to claim 9, wherein said oligosaccharide isselected from the group consisting of Gal Gal β(1-3)-GalNAc α(1-0)-, Galβ(1-3)-[GlcNAc β(1-6)]-GalNAc α(1-0)-, GlcNAc β(1-3)-GalNAc α(1-0)-,GlcNAc β(1-3)-[GlcNAc β(1-6)]-GalNAc α-(1-0)-, GalNAc α(1-3)-GalNAcα-(1-0)-, and Gal β(1-3)-[Gal β(1-6)]GalNAc α(1-0)-.
 11. The compositionaccording to claim 10, wherein said disaccharide is Gal β(1-3)GalNAc α-.12. The composition according to claim 10, wherein said monosaccharideis GalNAc.
 13. The composition according to claim 1, wherein saidmucin-type protein contains one or more asparagine amino acid residuesthat are fully N-linked glycosylated, partially N-linked glycosylated,or non-glycosylated at the amide group of asparagine, wherein saidasparagine is found in the sequence of amino acids Asn-X-Ser orAsn-X-Thr, wherein X is any amino acid other than proline and asparticacid.
 14. The composition according to claim 13, wherein said side chainis N-linked glycosylated with a carbohydrate selected from the groupconsisting of monosaccarides, disaccharides, oligosaccharides, andpolysaccharides.
 15. The composition according to claim 13, wherein theside chain is N-linked glycosylated with a hexasaccharide, GlcNAc3Man3.16. The composition according to claim 3, wherein said mucin-typepeptide is non-glycosylated.
 17. The composition according to claims 3or 16, wherein said mucin-type peptide, whether glycosylated or not, isan epitope associated with a mucin from malignant cells.
 18. Thecomposition according to claims 3 or 16, wherein said mucin-typepeptide, whether glycosylated or not, is an epitope associated with amucin from normal cells.
 19. The composition according to claims 1 or 3,wherein said mucin-type protein or mucin-type peptide is derived from acell surface coating of a gastrointestinal tract, eye, trachea, lung,salivary gland, sweat gland, breast, reproductive tract, pancreaticduct, gall bladder or urethra.
 20. The composition according to claim19, wherein said mucin-type protein or mucin-type peptide is derivedfrom a mammalian mucin.
 21. The composition according to claim 20,wherein said mammalian mucin is selected from the group consisting ofhuman, apes, monkey, rat, pig, dog, rabbit, cat, cow, horse, mouse, rat,and goat.
 22. The composition according to claims 1 or 3, wherein saidmucin-type protein or mucin-type peptide comprises a tandem repeatsequence selected from the group consisting of SEQ ID NOS:1-13.
 23. Thecomposition according to claim 3, wherein said mucin-type peptide is 5to 100 amino acids in length.
 24. The composition according to claim 1,wherein said mucin-type protein is greater than 100 amino acids inlength.
 25. The composition according to claims 1 or 3, wherein saidsolid support matrix comprises of an inorganic molecule, a polymer or acopolymer.
 26. The composition according to claim 25, wherein saidmatrix is an inorganic molecule selected from the group consisting of asilicate, alumina, hydroxyapatite, zeolite, germanate, phosphate, and amixture thereof.
 27. The composition according to claim 26, wherein saidinorganic molecule is a functionalized silicate.
 28. The compositionaccording to claim 27, wherein said functionalized silicate is selectedfrom the group consisting of 3-aminopropyl silica, 1-allyl silica,3-(3,4-cyclohexyldiol)propyl, 3-(diethylenetriamino)propyl, 4-ethylbenzenesulfonamide, 3-mercaptopropyl, propionyl chloride, 3-(2-succinicanhydride)propyl, and 3-(ureido)propyl.
 29. The composition according toclaim 25, wherein said solid support matrix is a polymer selected fromthe group consisting of agarose, dextran, polystyrene, polyvinylalcohol, polymethylacrylate, polymethylmethacrylate, and acrylamides.30. The composition according to claim 25, wherein said solid supportmatrix is a copolymer selected from the group consisting ofpolyethyleneglycol-co-polystyrene, polystyrene-co-divinylbenzene,sulfonated styrene-divinylbenzene, polyvinylchloride-co-vinylacetate,bis-acrylamide-azalactone, N-vinylpyrrolidone-co-divinylbenzene,polyethylene-co-butyl methacrylate, poly(lactic-co-glycolic acid),lignin-cresol copolymers, dextran-agarose copolymers, glycidylmethacrylate-ethylene dimethacrylate copolymers,poly(lactide-co-caprolactone) and polyacrylic copolymers.
 31. Thecomposition according to claims 1 or 3, wherein said amphiphilicmolecule comprises an epithelial cell membrane constituent.
 32. Thecomposition according to claims 1 or 3, wherein said amphiphilicmolecule is a phospholipid.
 33. The composition according to claims 1 or3, wherein said amphiphilic molecule is selected from the groupconsisting of phosphoglycerides, spingolipids, prostaglandins, saturatedfatty acids, unsaturated fatty acids and soaps.
 34. The compositionaccording to claims 1 or 3, wherein said amphiphilic molecule isphosphatidyl choline.
 35. The composition according to claim 1, whereinsaid mucin-type protein or mucin-type peptide is prepared by: (a)isolation from a biological source; (b) a recombinant DNA method; (c) asynthetic chemical route; or (d) a combination of any (a), (b), and/or(c).
 36. The composition according to claim 35, wherein recombinant DNAmethod comprises the step of expressing the mucin-type protein ormucin-type peptide from a host cell, wherein a nucleic acid moleculeencoding said mucin-type protein or mucin-type peptide has beenintroduced into the host cell.
 37. The composition according to claim36, wherein said nucleic acid molecule is a vector comprising a nucleicacid sequence encoding said mucin-type peptide or mucin-type protein.38. The composition according to claim 37, wherein the vector furthercomprises an expression control sequence operably linked to the nucleicacid sequence.
 39. The composition according to claim 37, wherein thenucleic acid sequence encodes for an amino acid sequence of a mucin-typeprotein or mucin-type peptide derived from a gastrointestinal tract. 40.The composition according to claim 36, wherein said host cell isselected from the group consisting of prokaryotic and eukaryotic cells.41. The composition according to claim 40, wherein said prokaryotic cellis an E. coli cell.
 42. The composition according to claim 40, whereinsaid eukaryotic cell is selected from the group consisting of yeast,insect and mammalian cells.
 43. The composition according to claim 42,wherein said eukaryotic cell is a mammalian cell.
 44. A method forpreparing a composition comprising a mucin-type protein or a mucin-typepeptide non-covalently immobilized to a solid support matrix, whereinsaid matrix is surface modified with an amphiphilic molecule, comprisingthe step of: mixing a solution comprising a mucin-type protein and/ormucin-type peptide with a solid support matrix that is surface modifiedwith an amphiphilic molecule under conditions in which the mucin-typeprotein and/or mucin-type peptide is immobilized to the matrix.
 45. Themethod according to claim 44, wherein the solution of mucin-type proteinor mucin-type peptide is a 10% (w/v) solution of protein/peptide in asolvent.
 46. The method according to 45, wherein the solvent is polar.47. The method according to 46, wherein the polar solvent is acetone orDPBS/Brij-35.
 48. The method according to claim 44, wherein said matrixis present in a ratio of 1:4 to 4:1 (w/v) to a 10% (w/v) solution ofprotein/peptide in a solvent.
 49. The method according to claim 44,wherein said mixing comprises the step of stirring the mixture in aclosed vial for 1 h at 298 K.
 50. The method according to claim 49,further comprising the step of washing the mixture multiple times with asolvent.
 51. The method according to claim 44, further comprising adrying step after said mixing step.
 52. The method according to claim51, wherein said drying step comprises evaporating said solvent in airor by rotoevaporation.
 53. The method according to claim 51, furthercomprising the step of packing a column for use in liquidchromatography, high performance liquid chromatography, or fastperformance liquid chromatography; coating a glass slide for thin layerchromatography; or coating a multi-well plate for HTS.
 54. A method forseparating one or more components of a mixture, comprising the steps ofcontacting the mixture with a chromatography medium comprising amucin-type protein, a mucin-type peptide, or a combination of thereof,wherein said mucin-type protein, mucin-type peptide or combinationthereof is immobilized to a solid support matrix, wherein said solidsupport matrix is surface modified with an amphiphilic molecule, underconditions in which one or more components of the mixture binds to thecomposition; and eluting the chromatography medium under conditions inwhich one or more components are separated from each other.
 55. Themethod according to claim 54, wherein the separation is performed bybatch chromatography.
 56. A method according to claim 54, wherein theseparation is performed via column chromatography.
 57. A method toestimate the permeability coefficient of a drug comprising the steps of:a) providing a chromatography medium, wherein said medium comprises amucin-type protein, mucin-type peptide, or a combination of thereofimmobilized to a solid support matrix, wherein solid support matrix issurface modified with an amphiphilic molecule; b) contacting a drug withsaid chromatography medium; c) measuring the degree of binding of thedrug on the chromatography medium; and d) estimating the drugpermeability coefficient.
 58. A method to estimate the permeabilitycoefficient of a drug comprising the steps of: a) providing achromatography medium, wherein said medium comprises a mucin-typeprotein, mucin-type peptide, or a combination of thereof immobilized toa solid support matrix, wherein solid support matrix is surface modifiedwith an amphiphilic molecule; b) contacting a drug with saidchromatography medium; c) recording a retention time of said drug; d)determining a membrane diffusion coefficient; e) determining a membraneequilibrium constant; f) determining a membrane thickness; and g)calculating an effective drug permeability.
 59. A method according toclaim 58, wherein a mobile phase is used, and wherein said mobile phaseis selected from the group consisting of acetone, DPBS with Brij-35 andethanol.
 60. A method according to claim 58, wherein said solvent isDPBS with Brij-35.
 61. A method according to claim 57, wherein said drugis in solution.
 62. A method according to claim 57, wherein the membranethickness is determined by confocal microscopy.
 63. The method accordingto claim 57 or 58, wherein more than one drug permeability coefficientsis measured.
 64. The method according to claim 63, wherein the drugpermeability coefficients are measured sequentially or simultaneously.65. The method according to claim 57 or 58, wherein the chromatographymediua comprises types and/or amounts of mucin-type protein, mucin-typepeptide or combinations thereof to mimic the mucus layer of the GItract, eye, trachea, lungs, salivary glands, sweat glands, breast,reproductive tract, pancreatic duct, gall bladder or urinary tract. 66.A method of comparing the relative permeability of one or more drugscomprising the steps of: a) providing a chromatography medium, whereinsaid medium comprises a mucin-type protein, mucin-type peptide, or acombination of thereof immobilized to a solid support matrix, whereinsolid support matrix is surface modified with an amphiphilic molecule;b) contacting a drug with said chromatography medium; c) measuring thedegree of binding of the drug on the chromatography medium; and d)comparing the degree of binding to that measured for one or more otherdrugs.
 67. The method according to claim 66, wherein the drug is onehaving an unknown drug permeability coefficient.
 68. The methodaccording to claim 66, wherein the drug is one having a known drugpermeability coefficient.
 69. The method according to claim 68, furthercomprising the step of calculating the permeability coefficient for thedrug of interest.
 70. A method of estimating drug permeability in two ormore physiological states comprising the steps of: a) providing a firstchromatography medium, wherein said medium comprises a mucin-typeprotein, mucin-type peptide, or a combination of thereof immobilized toa solid support matrix, wherein solid support matrix is surface modifiedwith an amphiphilic molecule and wherein said first chromatographymedium mimics the mucus layer of a first physiological state; b)providing a second chromatography medium, wherein said medium comprisesa mucin-type protein, mucin-type peptide, or a combination of thereofimmobilized to a solid support matrix, wherein solid support matrix issurface modified with an amphiphilic molecule and wherein said secondchromatography medium mimics the mucus layer of a second physiologicalstate; c) contacting a drug with said first and said secondchromatography media; d) measuring the degree of binding of the drug onthe first and second chromatography media; and e) comparing the degreeof binding on said first chromatography medium to that measured for saidsecond chromatography medium.
 71. The method according to claim 70,wherein said first physiological state is healthy tissue and said secondphysiological state is diseased tissue.
 72. The method according toclaim 71, wherein said first physiological state is normal epithelialtissue and said second physiological state is cancerous epithelialtissue.
 73. The method according to claim 70, wherein said firstphysiological state is a different developmental stage than said secondphysiological state.
 74. The method according to claim 73, wherein saidfirst and second physiological states are selected from an infant, achild, an adolescent, an adult and an elderly adult.
 75. A method toemulate an absorption process in a system comprising more than oneepithelial mucosal layer, comprising the steps of: a) providing a seriesof chromatography media, wherein said media comprises a mucin-typeprotein, mucin-type-peptide, or a combination of thereof immobilized toa solid support matrix, wherein solid support matrix is surface modifiedwith an amphiphilic molecule; wherein each of said media comprises adifferent amount and/or type of a mucin-type protein, mucin-type peptideor combination thereof, and wherein each of said media mimic thedifferent mucus linings of the system; b) contacting a drug with each ofsaid chromatography media; c) measuring the degree of binding of thedrug on each of said chromatography media; and d) comparing the degreeof binding to that measured for one or more other drugs.
 76. The methodaccording to claim 75, wherein said system is the digestive system. 77.A kit comprising mucin immobilized chromatography media and a standard.